Alzheimer's disease (AD) currently affects over 5 million people in the United States, with this number expected to rise dramatically (Hebert et al. (2013) Neurology 80:1778). AD is a progressive, insidious neurodegenerative disease of aging resulting in a gradual decline of cognitive function. AD progresses with a complex, multifactorial etiology, however there are two pathological entities present in AD that present targets for both diagnostic and therapeutic endeavors. These targets are insoluble extracellular amyloid beta (AB) plaques, and intracellular neurofibrillary tangles resulting from aggregates of hyperphosphorylated tau, a microtubule-associated protein. While the insoluble deposits of AB plaques serve as a marker for AD, multiple genetic and biochemical lines of evidence support the hypothesis that the Alzheimer's causative agent is the soluble, oligomeric form of the AB peptide (Viola and Klein (2015) Acta Neuropathol. 129:183). AB plaques are generated from the aggregation of soluble AB peptides formed when y and 3 secretases (proteases) cleave the amyloid precursor protein (APP), a protein constitutively expressed on the plasma membrane of neurons (Zhang et al. (2011) Mol. Brain 4:3). Because considerable brain damage due to amyloid plaque deposition typically occurs 10-20 years before a definitive diagnosis of dementia can be made (Sperling et al. (2011) Alzheimers Dement. 7:280 and Bateman et al. (2012) N. Eng. J. Med. 367:795). It is imperative to develop methods capable of detecting the Alzheimer's disease process at the earliest possible stage. Currently the only definitive diagnostic of AD is applied post mortem, and clinical diagnosis tools are largely limited to memory testing and PET imaging. An alternative accessible and non-invasive method capable of the early detection of AB levels in the brain could be useful in identifying disease risk, facilitating clinical trials, tracking disease progression, and guiding therapies.
Imaging techniques utilizing radiolabeled positron emission tomography (PET) or single photon emission computed tomography (SPECT) that bind to AB peptides in amyloid plaques have the potential to directly assess amyloid burden (Klunk et al. (2004) Ann. Neurol. 55:306), but suffer from limited availability, high cost, and a reliance on short-lived radioisotopes. In this regard, a detection method based on Magnetic Resonance Imaging (MRI) for AD risk is highly desirable. MRI can provide advantages over PET and SPECT that include the use of non-radio-active probes, an increased resolution, and the ability to define anatomic details that can be used to quantify the amyloid plaque (Huddleston and Small (2005) Nat. Clin. Pract. Neurol. 1:96).
MRI can detect deposits of amyloid plaques when a sufficient amount of endogenous iron is associated with the plaques (Vanhoutte et al. (2005) Magn. Reson. Med. 53:607). The presence of metals, in particular the iron in the plaques, generates an accelerated T2* relaxation rate and negative contrast at regions high in iron-rich AB (Vanhoette et al. (2005) and Jack et al. (2007) Neuroscientist 13:38). However, a method dependent on endogenous iron is hindered since T2* contrast might originate from different sources, such as heme iron present in red blood cells or non-heme iron present in cerebral tissue (Jack et al. (2007) and Gelman et al. (2001) J. Neurochem. 45:71). In addition, some regions of the brain contain iron-poor AB depositions (Vanhoutte et al. (2005) and Ghribi et al. (2006) J. Neurochem. 99:438), making accurate evaluation of total AB load by iron-induced contrast difficult.
While iron-rich amyloid plaque provides some negative contrast in MRI (Jack et al. (2007) and Wadghiri et al. (2012) Methods Mol. Biol. 849:435), the lack of signal from iron-poor amyloid limits the significance of the approach for a general assessment of AB levels in the brain (Adlard et al. (2014) Front Neurosci. 8:327). Towards the goal to diagnose AD using MRI, various agents that target AB to generate enhanced contrast in MRI have been created. For example, magnetic nanostructures doped with superparamagnetic iron have been tethered to antibodies directed against the oligomeric form of AB to generate contrast in the brains of AD mice (Viola et al. (2015) Nat. Nanotechnol. 10:91). In addition, several Gd(III)-based approaches have explored conjugating a metal chelator to either AB itself (Wadghiri et al. (2012) and Poduslo et al. (2002) Neurobiol. Dis. 11:315), or to an antigen-binding fragment directed against AB (Ramakrishnan et al. (2008) Pharm. Res. 25:1861). Small molecules with affinity for AB assemblies have also been combined with Gd(III) chelators to produce contrast at AB, however addition of the chelated Gd(III) to the targeting molecule diminishes amyloid binding and blood-brain barrier permeability (Bort et al. (2014) Eur. J. Med. Chem. 87:843). While metal-containing and/or antibody-directed probes show promise, concerns and limitations drive the need for alternative small molecule agents. For example antibody-directed agents may increase inflammatory cascades (Fuller et al. (2015) 130:699), and use of immunoglobin-conjugated Gd(III) agents have low transport in the parenchyma of the brain (Wadghiri et al. (2012)). In addition, use of chelated Gd(III) is contraindicated in many patients with kidney or liver disease (Khawaja et al. (2015) Insights Imaging 6: 553). More importantly, the recent evidence that gadolinium is sequestered in the brain many years after contrast-enhanced MRI (Karabulut (2015) Diagn. Interv. Radiol. 21:269) provides an impetus to explore gadolinium-free imaging options for both immunoglobulin-as well as small molecule-based amyloid specific ligands.
One option for creating such ligands is through the attachment of a spin label such as nitroxide. Spin labeled compounds have been shown to interact with AB peptide oligomers and to demonstrate potential therapeutic value (Altman et al. (2015) Biochim. Biophys. Acta 2854:1860; Petrlova et al. (2012) PLoS One 7:e35443; and Hong et al. (2010) Neurobiol. Aging 31:1690). Other work has demonstrated the use of labeled thioflavin compounds to study fibril redox state changes with fluorescence and electron spin resonance (Mito et al. (2011) Chem. Commun. 47:5070). However, the use of nitroxide spin labels in MRI applications has been limited due to a demonstrated weakness in relaxivity (Maliakal et al. (2003). J. Phys. Chem. A 107:8467; Winalski et al. (2008) Osteoarthritis Cartilage 16:815; and Rajca et al. (2012) J. Am. Chem. Soc. 134:15724). In addressing this limitation, the present invention surprisingly meets the need for accessible and non-invasive methods of amyloid imaging as well as other needs.